1. Field of the Invention
The present invention relates to antibacterial peptides and, more particularly, to antibacterial peptides which are partial peptide based upon the amino acid sequences of a guinea-pig-derived antimicrobial polypeptide and partially substituted forms of this peptide. The present invention also relates to antimicrobial agents; to inhibitors for inhibiting binding of lipopolysaccharide to a cell (hereinafter referred to as lipopolysaccharide-cell-binding inhibitor); and to a drugs such as a bacterial-infection-treating agents or endotoxin-shock suppressants, each employing the antibacterial peptides as an active ingredient.
2. Background Art
In the present specification, the following abbreviations are used.
CAP11: cationic antibacterial polypeptide of 11 kDa
E. coli: Escherichia coli 
FCS: fetal calf serum
HPLC: high performance liquid chromatography
LPS: lipopolysaccharide, also called endotoxin
MRSA: methicillin-resistant Staphylococcus aureus 
MSSA: methicillin-sensitive Staphylococcus aureus 
PBS: phosphate-buffered physiological saline
S. aur: Staphylococcus aureus 
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
CAP11 is an antibacterial polypeptide found in guinea-pig neutrophils and is known to be a homodimer consisting of peptides that are linked via a cysteine disulfide bond (S—S bond) with each peptide having 43 amino acid units (Yomogida, S., et al., Archives of Biochemistry and Biophysics, Vol. 328, p. 219-226 (1996)). CAP11 is known to exhibit a potent antibacterial effect to gram-positive and gram-negative bacteria and is known to neutralize bioactivity of gram-negative bacteria LPS (Nagaoka, I. et al., Inflammation Research, Vol. 49, p. 73-79 (2000) and Nagaoka, I. et al., Journal of Immunology, Vol. 167, p. 3339-3338 (2001)).
U.S. Pat. No. 6,040,291 issued to Hirata (hereafter “Hirata '291”) discloses a partial peptide of CAP18 (cationic antibacterial polypeptide of 18 kDa) having antimicrobial and LPS-binding activities.
If the aforementioned effects of CAP18 or CAP11 are obtained from a peptide of smaller size, and if its activity is higher than known partial peptide of CAP18 or CAP11, antibacterial agents and various drugs showing strong activities could be produced more speedily and easily at a lower cost, while facilitating quality control of the products and other operations. Also, if such partial peptides exceed the efficacy of CAP11 or the partial peptides of CAP18, the benefits would be even greater.
Hirata '291 discloses enhancements to partial peptides of CAP18 by substituting certain amino acids naturally occurring in the CAP11 partial peptide sequence so as to effect a different balance between hydrophobic and hydrophilic amino acids. Hirata '291 proposed that the reason why the peptides of its disclosure have high antimicrobial activity, the high endotoxin (LPS)-binding activity and the high endotoxin (LPS)-neutralizing activity is that the peptide in the LPS-binding domain of human-derived CAP18 has an alpha-helix structure, which, when projected in its axial direction (depicted as a helical wheel in FIGS. 1 and 3 of Hirata '291) reveals a hydrophilic portion (i.e., a portion which is rich in a hydrophilic amino acid residue (basic amino acid residue) such as arginine and lysine) and a hydrophobic portion (i.e., a portion which is rich in a hydrophobic amino acid residue such as phenylalanine, leucine and isoleucine). Hirata presumed that the hydrophilic portion of the peptide binds ionically to a portion of the phosphate group of the lipid A portion of LPS, and the hydrophobic portion of the peptide hydrophobically binds to the fatty acid portion of the lipid A, resulting in exhibition of the antimicrobial activity and the LPS-neutralizing activity.
Accordingly, Hirata '291 proposed that substitution of specified amino acid residues at specified positions would alter the balance between the hydrophilic portion and the hydrophobic portion (FIGS. 1 and 3) and this change would be associated with an increase in the antimicrobial activity, the LPS-binding activity, and LPS-neutralizing activity.
While Hirata '291 disclosed the concept that designing an amino acid sequence of a partial peptide of CAP18, by taking into consideration the balance between the hydrophilic portion and the hydrophobic portion in the helical wheel when the alpha-helix structure of the peptide is projected in its axial direction, will increase the antimicrobial activity, the LPS-binding activity, and the LPS-neutralizing activity, it disclosed only substitutions which increased the hydrophobic aspect of the natural partial peptide (of CAP18) (see FIGS. 2 and 4 of Hirata '297).
In other words, while it appears that the substituted partial peptides according to Hirata '291 are successful to accomplish the stated goals of Hirata '291, the “balance” of the substitutions disclosed in Harata '291 favored a hydrophobic character. Hirata '291 does not disclose making substitutions to a natural peptide amino acid sequence to increase bi-lateral symmetry between the hydrophobic and hydrophilic character of an amino acid sequence of a peptide used as an antimicrobial agent. It follows that Hirata '297 also did not disclose synthesizing a peptide for antimicrobial use having substantial bi-lateral symmetry when viewed axially as a helical wheel, or the enhanced activity of such a peptide.